Enabling programmable dynamic DNA chemistry using small-molecule DNA binders

The binding of small molecules to the double helical structure of DNA, through either intercalation or minor groove binding, may significantly alter the stability and functionality of DNA, which is a fundamental basis for many therapeutic and sensing applications. Here, we report that small-molecule DNA binders can also be used to program reaction pathways of a dynamic DNA reaction, where DNA strand displacement can be tuned quantitatively according to the affinity, charge, and concentrations of a given DNA binder. The binder-induced nucleic acid strand displacement (BIND) thus enables innovative technologies to accelerate the discovery and characterization of bioactive small molecules. Specifically, we demonstrate the comprehensive characterization of existing and newly discovered DNA binders, where critical parameters for binding affinity and sequence selectivity can be obtained in a single, unbiased molecular platform without the need for any specialized equipment. We also engineer a tandem BIND system as a high-throughput screening assay for discovering DNA binders, through which 8 DNA binders were successfully discovered from a library of 700 compounds.

Fluorescence Indicator Displacement (FID) Assay. Endpoint fluorescence measurement was used in FID assay for the high throughput screen (HTS) of new binders. Briefly, 20 nM CP was mixed 1 µM of EB in 1 x TE buffer and then incubated at 37 °C for 2 hours.
To this reaction mixture, a candidate compound at final concentration of 30 µM was added.
After another incubation at 37 °C for 2 hours, endpoint fluorescence was measured using BioTek Cytation 5 Multimode Microplate reader at excitation/emission wavelength at 522 nm/593 nm. The fluorescence signal in each well was normalized using a positive control containing 20 nM CP and 1 µM of EB and a negative control containing 20 nM CP in 1 x TE buffer. Chemdraw software, and .pdb files of DNA were obtained through PDB. In the preliminary preparations for the molecular docking, the binders were successively hydrogenated, detected root, chosen torsion and finally converted into PDBQT files, while DNAs were similarly hydrogenated, removed water molecules, calculated gaseiger, assigned AD4 type, and finally converted to PDBQT files. The grid box was uniformly set to contain the entire DNA structure. During the docking process, the Lamarckian genetic algorithm was used for binder-DNA docking. Among them, the maximum number of energy evaluations was set to 2,500,000. The maximum number of generations was 27,000. The rate of gene mutation was 0.02. The rate of crossover was 0.8. Maximum number of top individuals that automatically survive was 1. Finally, we comprehensively evaluated and selected the optimal binding conformation of binder and DNA according to the stable binding energy, which were visualized with Pymol software.

S8
Cytotoxicity study (MTT assay) of S20 with three neoplastic cell lines. Three cell lines Hela (ATCC, CCL-2), HepG2 (ATCC, HB-8065), and A549 (ATCC, CCL-185) were purchased from the American Type Culture Collection (ATCC, Manassas, VA). Cells were cultured in Dulbecco's modified Eagle medium containing 10% fetal bovine serum (Gibco), penicillin (100 U/mL), and streptomycin (100 μg/mL). Cells were seeded into 96-well plates (BIOFIL, catalog number TCP-011-096) at 1 × 10 4 cells per well and incubated for 12 h at 37 °C in 5% CO2 to facilitate attachment. Cells were treated with different concentrations of compounds in DMEM with 10% FBS and then incubated for 24 h. Cells with no compounds added served as controls. After incubation, old media was removed, and cells were washed with PBS once before cell media was replaced with 120 μL of fresh media with MTT (0.5 mg/mL). Cells were incubated for another 1.5 h at 37 °C in 5% CO2.

S2. Theoretical Modeling and Fitting Thermodynamic Model
A theoretical model for BIND was established by considering the strand displacement to be an SN1 reaction in the absence of the binder or the binder concentration was below the critical binder concentration (CBC). When the binder concentration was greater than CBC, the reaction was considered to go through an SN2 strand displacement reaction pathway.
To quantitatively profile BIND, we established a workflow for extracting critical thermodynamic parameters by combining experimental measurement and theoretical fitting (Fig. S2). We first derived the initial values of and in the absence of any binder by fitting the experimental data. Equilibrium constants when binder concentration equals to CBC were also determined. values in the presence of binders with concentrations below CBC were then determined by fitting experimental data in a dissociation reaction of CP in the presence of binders but absence of the invader I (Fig.   S1h). and the overall strand displacement equilibrium 1 could then be determined by fitting the strand displacement reaction using the SN1 reaction mechanism and experimentally determined reaction yield in the presence of a given concentration of binder.
By further determining the Gibbs free energy of the reaction using the equilibrium constant ( Fig. S2), we observed a linear relationship between ∆ 1 and the concentrations of DNA binders (Fig. S3). The similar mathematical treatment at the SN2 region also revealed a linear relationship of ∆ 2 against binder concentration above CBC (Fig. S3).